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Originally published In Press as doi:10.1074/jbc.M408187200 on September 15, 2004

J. Biol. Chem., Vol. 279, Issue 47, 48787-48793, November 19, 2004
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EmrE, a Multidrug Transporter from Escherichia coli, Transports Monovalent and Divalent Substrates with the Same Stoichiometry*

Dvir Rotem{ddagger} and Shimon Schuldiner§

From the Alexander Silberman Institute of Life Sciences, Hebrew University of Jerusalem, 91904 Jerusalem, Israel

Received for publication, July 20, 2004 , and in revised form, September 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Multidrug transporters recognize and transport substrates with apparently little common structural features. At times these substrates are neutral, negatively, or positively charged, and only limited information is available as to how these proteins deal with the energetic consequences of transport of substrates with different charges. Multidrug transporters and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites, and human cancer cells. Understanding how these efflux systems handle different substrates may also have practical implications in the development of strategies to overcome the resistance mechanisms mediated by these proteins. Here, we compare transport of monovalent and divalent substrates by EmrE, a multidrug transporter from Escherichia coli, in intact cells and in proteoliposomes reconstituted with the purified protein. The results demonstrated that whereas the transport of monovalent substrates involves charge movement (i.e. electrogenic), the transport of divalent substrate does not (i.e. electroneutral). Together with previous results, these findings suggest that an EmrE dimer exchanges two protons per substrate molecule during each transport cycle. In intact cells, under conditions where the only driving force is the electrical potential, EmrE confers resistance to monovalent substrates but not to divalent ones. In the presence of proton gradients, resistance to both types of substrates is detected. The finding that under some conditions EmrE does not remove certain types of drugs points out the importance of an in-depth understanding of mechanisms of action of multidrug transporters to devise strategies for coping with the problem of multidrug resistance.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Multidrug transporters (MDTs)1 and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites, and human cancer cells (1–3). Phylogenetic studies show that these efflux systems are associated with five superfamilies of transporters (4). One of these includes a family of small multidrug resistance (SMR) conferring proteins. The SMR family consists of small hydrophobic proteins of ~100 amino acid residues with four transmembrane {alpha}-helical spanners (5–7). These proteins remove cationic drugs from the cytoplasm using a drug/H+ antiport mechanism (5–7).

Genes coding for SMR proteins have been identified in many eubacteria and in some Archaea (8, 9). The most extensively characterized SMR protein is EmrE, from Escherichia coli. The protein has been characterized, purified, and reconstituted in a functional form (10). High-affinity substrate binding has been established as a reliable and sensitive assay for activity of the detergent-solubilized transporter (11–13).

Glu-14, the only membrane-embedded charged residue, is highly conserved in the SMR family (8). This residue has an unusually high pK and is an essential part of the binding domain shared by substrates and protons (11–13). The occupancy of the binding domain is mutually exclusive, and as such this provides the molecular basis for coupling of substrate and proton fluxes. Direct measurements of substrate-induced release of protons in a detergent-solubilized EmrE shows the stoichiometry of the release is almost 1 proton per monomer. The findings demonstrate that the only residue involved in proton release is Glu-14 and that all the Glu-14 residues in the EmrE functional oligomer participate in proton release (14).

MDTs transport substrates with apparently little common structural features. At times these substrates are neutral, negatively, or positively charged (15–18). This poses intriguing mechanistic challenges such as binding of substrates with different charges to a common binding domain. Some clues for the molecular basis of multidrug recognition are emerging from the structural studies of transcription factors that regulate expression of MDTs (19). Only limited information is available as to how the MDTs deal with transport of substrates with different charges. The gradient that ion-coupled transporters can generate depends on the number of ion molecules transported per substrate molecule (stoichiometry) and on the charge of the substrate. In the case of MdfA, an E. coli multidrug transporter of the major facilitator superfamily, it was shown that it exchanges neutral compounds as chloramphenicol and thiamphenicol and monovalent cationic substrates with the same stoichiometry, and as a result the driving forces for both types of substrates are different (20).

In this study, we compare transport of monovalent and divalent substrates by EmrE in intact cells and in proteoliposomes reconstituted with the purified protein. In intact cells, under conditions where the only driving force is the electrical potential, EmrE confers resistance to monovalent substrates but not to divalent ones. In the presence of a proton gradient, resistance to both types of substrates is detected. In proteoliposomes reconstituted with purified EmrE only proton gradients were able to drive transport of both divalent and monovalent substrates, whereas the electrical potential drove transport of monovalent substrates. The results demonstrated that whereas the transport only of monovalent substrates involves charge movement (i.e. electrogenic), the transport of divalent substrate does not (i.e. electroneutral). Together with previous results, these findings suggest that an EmrE dimer exchanges two protons per substrate molecule during each transport cycle. The findings that under some conditions EmrE is ineffective in removing certain types of drugs imply that an in-depth study of mechanisms of action of MDTs may help devising strategies for coping with the problem of multidrug resistance.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial Strains and Plasmids—E. coli JM109 (21) and TA15 strains (22) are used throughout this work. The plasmids used are pT7–7 (23) derivatives for wild type with (EmrE-His (11)) or without (10) His6 tag.

Resistance to Toxic Compounds—Resistance to toxic compounds methyl viologen (MV2+), ethidium, or acriflavine was tested by measuring optical density of 0.3-ml cultures after overnight growth of JM109 cells bearing plasmids pT7–7 with or without the EmrE gene. Growth was at 37 °C in a 1.2-ml-square storage plate (96 wells, Abgene, Surrey, UK). LB medium containing 50 µg/ml ampicillin and 30 mM Bis-Tris propane was titrated to the indicated pH with HCl. Bis-Tris propane was added to maintain a constant pH during growth as described previously (24).

Overexpression, Purification, and Reconstitution of EmrE-His— Overexpression and purification of EmrE was performed essentially as described previously (14). For reconstitution, purified EmrE-His was incubated with nickel-nitrilotriacetic acid beads (Qiagene GmbH, Hilden, Germany) for 1 h at 4 °C. The beads were washed once with buffer containing 150 mM NaCl, 15 mM Tris-Cl, pH 7.5 (sodium buffer) with 0.08% n-dodecyl-{beta}-D-maltoside (Glycon Biochemicals, Luckenwalde, Germany), 15 mM {beta}-mercaptoethanol, and 3x in sodium buffer containing 1% n-octyl-{beta}-D-glucopyranoside (Glycon Biochemicals, Luckenwalde, Germany). EmrE-His was eluted from the beads with sodium buffer containing 1% n-octyl-{beta}-D-glucopyranoside and 200 mM imidazole. The supernatant was mixed with a solution containing 25 mg/ml E. coli phospholipids (Avanti, Inc., Alabaster, AL), 150 mM NaCl, 15 mM Tris-Cl, pH 7.5, 1% n-octyl-{beta}-D-glucopyranoside. The amount of protein mixed with the phospholipids is indicated in the specific experiments. To generate {Delta}pH, after sonication the mixture was diluted 27-fold into NH4 buffer containing 190 mM NH4-Cl, 15 mM Tris-Cl, pH 7.5, and 1 mM dithiothreitol, incubated for 20 min at 25 °C, and then centrifuged at 240,000 x g for 90 min. Proteoliposomes were resuspended in NH4 buffer, frozen in liquid air, and kept at –70 °C.

To generate {Delta}{psi}, the proteoliposomes were formed by dilution into 70 mM sodium thiocyanate, 120 mM NaCl, 20 mM Tris-Tricine buffer, pH 7.5 and 1 mM dithiothreitol (thiocyanate buffer). They were then centrifuged, resuspended and frozen as above. Before the assay, the proteoliposomes suspension was thawed and sonicated in a bath-type sonicator for a few seconds until clear.

{Delta}pH-driven Methyl Viologen Uptake Assay—Uptake of [14C]methyl viologen into proteoliposomes was assayed essentially as described in Yerushalmi et al. (10). 3 µl of the NH4 containing proteoliposomes were diluted into 200 µl of an ammonium-free solution. The latter contained 20 µM [14C]methyl viologen (32 nCi/assay), 140 mM KCl, 10 mM Tricine, 10 mM Tris, and 5 mM MgCl2 (final pH 8.5) (KCl buffer). At given times the reaction was stopped by dilution with 2 ml of the same ice-cold solution. The samples were filtered through Millipore filters (0.22 µm) and washed with an additional 2 ml of solution. The radioactivity on the filters was measured by liquid scintillation. In each experiment the values obtained in a control reaction with 15 µM nigericin were subtracted from all experimental points. This background was no more than 10% of most experimental values. The reaction was measured in duplicates. Each experiment was performed at least twice.

{Delta}pH-driven Tetraphenylphoshonium (TPP+) Uptake Assay—3 µl of NH4 containing proteoliposomes were diluted into 200 µl of KCl buffer that contained 10 nM [3H]TPP+ (60 nCi/assay). Because of the relatively high nonspecific binding of [3H]TPP+ to the filters, the reaction was slightly modified so that it was stopped by centrifugation (452 x g, 1 min) through columns (2-ml disposable syringes) containing Sephadex G-50 fine, preswollen in KCl buffer and packed by centrifugation (113 x g, 1 min). The radioactivity was measured by liquid scintillation. In each experiment, a control reaction was carried out where no gradient was generated (dilution into ammonium buffer). The values obtained in such a reaction were subtracted from all experimental points. The reactions were carried out in duplicates. Each experiment was performed at least twice.

For examining the effect of N,N'-dicyclohexylcarbodiimide (DCCD) on [3H]TPP+ uptake, proteoliposomes were incubated with 0.3 mM DCCD for 30 min before the uptake assay. Drug gradients were calculated using intraliposomal volumes determined in a separate experiment as follows: proteoliposomes were sonicated in the presence of 2 mM [14C]methyl viologen (32.3 mCi/mmol). A sample (3 µl) was diluted with 2 ml of the same ice-cold solution used in the transport experiments, filtered through Millipore filters (0.22 µm), and washed with an additional 2 ml of solution. From the radioactivity associated with the proteoliposomes an internal volume of 0.2 µl per sample was calculated. To prevent significant leakage this manipulation was performed with proteoliposomes reconstituted with EmrE E14C, an inactive mutant (10).

TPP+ Binding Assay—NH4 containing proteoliposomes were diluted into 200 µl of a buffer containing 10 nM [3H]TPP+ (60 nCi/assay), 190 mM NH4Cl, and 43 mM Tris-Cl (final pH 8.5). The reactions (200 µl) were stopped by centrifugation (452 x g, 1 min.) through columns containing Sephadex G-50 fine, preswollen in the same buffer, and packed by centrifugation (113 x g, 1 min.). The radioactivity was measured by liquid scintillation.

{Delta}{psi}-Driven Uptake Assays—{Delta}{psi} driven uptake assays of MV2+ and TPP+ were performed as the {Delta}pH-driven uptake assays except that the inner and outer solutions differed in composition; 3 µl of proteoliposomes formed in thiocyanate buffer were diluted into 200 µl of a solution containing 10 nM [3H]TPP+ or 41 µM [14C]MV2+, 190 mM KCl, and 20 mM Tris-Tricine buffer, pH 7.5. As a control the proteoliposomes were diluted into a buffer of composition identical to the one in their interior. Results were essentially identical when the membrane potential was generated by a valinomycin-induced K+ diffusion potential in a medium with no chloride as described in Ref. 20.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The Resistance That EmrE Confers against Monovalent and Divalent Substrates Is Affected Differently by Changes in the External pH—E. coli cells that overexpress EmrE display resistance to a wide variety of toxic lipophilic cations, monovalents such as ethidium and acriflavine, and divalents such as MV2+ (10). The growth of E. coli cells carrying plasmids with or without the emrE gene in LB medium containing monovalent or divalent lipophilic cations was compared under various pH conditions (Fig. 1). In the absence of drugs, cells harboring either plasmid grew equally well in the pH range of 7.0–8.4 (data not shown). As expected when the monovalent substrates 125 µM ethidium or 62 µM acriflavine were added to the medium, only cells expressing EmrE were able to grow. At these drug concentrations growth was practically identical in the pH range tested. In contrast, the resistance that EmrE confers against 62 µM of the divalent substrate MV2+ is drastically reduced as the pH increases, and it is abolished above pH 7.6.



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  		FIG. 1.
Resistance to toxic compounds at different pH. Resistance to toxic compounds acriflavine (62 µM) (A), ethidium (125 µM) (B), or methyl viologen (62 µM) (C) was tested after overnight growth of JM109 cells bearing plasmids pT7-7 with (black squares) or without (white squares) the EmrE gene. Growth was for 14–16 h at 37 °C in LB medium containing 50 µg/ml ampicillin and 30 mM Bis-Tris propane titrated to the indicated pH with HCl.

 
To further explore the nature of this different sensitivity to pH, a more detailed study was carried out and the IC50 values of the three compounds were determined at the various pH values. The findings are illustrated for two pHs in each case (Fig. 2, A–C) and summarized in Fig. 2, right panels. Ethidium and acriflavine markedly inhibited growth at pH 7.0 and pH 8.4 with a slightly higher potency (about 5-fold) at the more alkaline pH (Fig. 2, A and B, gray lines). At the two pH values, EmrE significantly protected against the toxic effects of both compounds (black lines). In the case of MV2+, the difference between the two pH values is more striking. MV2+ was 15 times more potent at pH 8.4 than at pH 7.0 (Fig. 2C, gray lines). The protection conferred by EmrE at pH 8.4 was only slightly evident at the low concentration (black lines). Moreover, at this pH, EmrE induces a reproducible and significant sensitization at the higher concentrations.



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  		FIG. 2.
Determination of IC50 values of monovalent and divalent substrates. Growth of cells bearing plasmids pT7–7 with (black lines, squares) or without (gray lines, triangles) the EmrE gene at different pH conditions at different concentrations of acriflavin (A), ethidium (B), or methyl viologen (C) was tested. Growth was at 37 °C in LB medium containing 50 µg/ml ampicillin, 30 mM Bis-Tris propane titrated to the indicated pH with HCl, and different concentrations of the drugs. The experiment was repeated four times. The results of experiments at pH 7.0 (solid lines, black symbols) and pH 8.4 (dashed lines, white symbols) are presented in the left panel for illustration. Each curve is result of a fit obtained using Origin 7.0 software (OriginLab, North-ampton, MA). The IC50 values from all the pH values tested are summarized in the right panel. At the pH values above 7.8 and indicated with a * there is a slight EmrE-dependent resistance at low concentrations of MV2+, and EmrE-dependent sensitization at high concentrations are as shown for pH 8.4 in the left panel. IC50 values were calculated with Origin 7.0 software.

 
In Fig. 2, right panels, we can see the systematic decrease of IC50 values for all of the substrates tested at the high pH values. However, whereas the drop in IC50 for ethidium and acriflavine in the presence of EmrE was about 8- and 4-fold, respectively, in its absence there was also an increased toxicity of the compounds, and therefore the protection was still highly significant even at the high pH. On the other hand, for MV2+ IC50 in the presence of EmrE decreased 114-fold in the range between 7.0 and 8.4. In addition, the degree of protection was practically undetectable at the high pH values.

Increased sensitivity at higher pHs could be caused by several factors, including higher sensitivity of target and increased uptake and decreased extrusion by EmrE. The latter could be caused by changes in the driving forces for extrusion or in its components. The internal pH of E. coli cells is kept in the range 7.6–7.9 (25). As a result, as external pH is raised, {Delta}pH (inside alkaline) drops and even becomes inverted (inside acid) above this pH, whereas {Delta}{psi} (inside negative) increases (25). This raises the hypothesis that EmrE can only utilize {Delta}pH to extrude MV2+ from the cells, suggesting the possibility that transport is electroneutral. Above pH 7.8–8.0, only compounds that are electrogenically transported out of the cell can be removed. In contrast, at high pH, without a significant driving force EmrE may facilitate the entry of compounds that are transported electroneutrally. This can explain the higher sensitivity to MV2+of cells that express EmrE at high pH (Fig. 2C). Therefore, a detailed study of the effect of the components of the proton electrochemical gradient on transport of monovalent and divalent substrates was carried out.

EmrE Catalyzes Uptake of TPP+ into Proteoliposomes— EmrE catalyzes {Delta}pH-driven uptake of MV2+ and ethidium into proteoliposomes (10). Transport of MV2+ can be measured rapidly and quantitatively using radiolabeled substrate (10). Transport of ethidium is followed using an assay based on changes of fluorescence of ethidium upon uptake to proteoliposomes loaded with nucleic acids. This is a convenient assay but only qualitative. Therefore it was necessary to introduce a new assay and we chose to study transport of TPP+, a compound that binds to detergent-solubilized EmrE with nanomolar affinity (11). We chose TPP+ because of its high affinity and because of the knowledge we have accumulated on its interaction with EmrE (6, 7). In these experiments, the driving force was a pH gradient generated by the ammonium diffusion gradient obtained upon dilution of the proteoliposomes prepared in NH4Cl medium into media in which the ammonium was replaced by KCl (10). A time-dependent accumulation of [3H]TPP+ was observed in proteoliposomes reconstituted with EmrE (Fig. 3A, black squares). When the pH gradient was abolished by dilution into a medium identical in composition to that inside the proteoliposome, a small rapid association of TPP+ to the proteoliposomes was observed (Fig. 3A, white squares). As shown in Fig. 3B, other substrates of EmrE, like MV2+, ethidium, and acriflavine inhibit the [3H]TPP+ uptake in a concentration range expected from their kinetic properties. In addition DCCD, a carbodiimide that is known to react with carboxyls and inhibits MV2+ uptake and TPP+ binding by EmrE (26), also inhibits transport of TPP+ (Fig. 3A, inset). The protein concentrations used in these experiments are at least four times lower than those used to measure MV2+ uptake. When higher concentrations are used, the fraction of TPP+ associated with the proteoliposomes in the absence of a pH gradient increases with the increase in amount of protein (Fig. 4). The gradients of TPP+ generated in the presence of are practically identical at all protein concentrations, but they become more difficult to detect. At the highest protein concentration used (750 ng) all the TPP+ is bound to EmrE, and therefore no transport can be detected. We conclude that the TPP+ associated with the proteoliposomes in the absence of a pH gradient is bound to EmrE because the binding is inhibited by other substrates (Fig. 4, inset) or by pretreatment of the protein with DCCD (Fig. 3A, inset) (26). In addition, although not shown, binding is not observed in liposomes with no protein or proteoliposomes prepared with E14C-EmrE, an inactive mutant (11). A more detailed study comparing the binding properties of TPP+ to the membrane associated and the detergent solubilized protein is now being carried on. Based on the results that demonstrated the ability of EmrE to catalyze uptake of [3H]TPP+ and [14C]MV2+ into proteoliposomes, we can compare the energetics of the uptake of monovalent and divalent substrates.



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  		FIG. 3.
TPP+ uptake activity into proteoliposomes reconstituted with purified EmrE. Purified EmrE was reconstituted essentially as described under "Experimental Procedures." A, ammonium-loaded EmrE proteoliposomes (3 µl) were diluted into the same ammonium medium (white squares) or into an ammonium-free medium (black squares) containing 20 nM [3H]TPP+. At the indicated time the proteoliposomes were separated from the medium by filtration on Sephadex G50 columns and radioactivity incorporated was measured. The inset shows [3H]TPP+ uptake (10 nM) measured after 2 min with or without an ammonium gradient after pretreatment of proteoliposomes with 300 µM DCCD for 30 min before the uptake assay. B, [3H]TPP+ uptake (1 min) measured in the presence of substrates of EmrE at the concentrations specified in the figure.

 



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  		FIG. 4.
Reconstituted EmrE binds TPP+. Proteoliposomes were reconstituted with the indicated amount of purified EmrE and loaded with ammonium. They were then diluted (3 µl) into an ammonium-free medium (black squares) or ammonium medium (white squares) containing [3H]TPP+. After 2 min, proteoliposomes were filtered on Sephadex G50 columns, and radioactivity incorporated was measured. Inset, [3H]TPP+ binding to ammonium-loaded EmrE proteoliposomes (3 µl) was measured in the presence of substrates of EmrE as specified in the figure.

 
{Delta}{psi} Drives EmrE-mediated Uptake of TPP+ but Not of MV2+—Depending on the proton/substrate stoichiometry of the reaction and the charge on the substrate, exchange of protons with a substrate by antiporters may result in a net movement of electric charges (electrogenic transport) or no net movement of charges (electroneutral transport). Electrogenic transport will be driven by both components ({Delta}{psi} and {Delta}pH) of the proton electrochemical gradient (), but when transport is electroneutral, {Delta}pH is the primary driving force (27, 28). To determine whether EmrE can utilize {Delta}{psi} as the driving force for uptake of substrates, we used proteoliposomes where {Delta}{psi} (positive inside) was generated by imposing a gradient of SCN–, a permeant anion (SCN–in> SCN–out). The results shown in Fig. 5 demonstrate EmrE-catalyzed uptake of the monovalent cation TPP+ into proteoliposomes when {Delta}{psi} (positive inside) is generated (filled squares). TPP+ uptake was abolished with DCCD, a carbodiimide that inhibits TPP+ binding to the protein (data not shown). The TPP+ concentration gradient generated by EmrE about 60-fold is almost identical to the predicted accumulation expected from the SCN– gradient generated by dilution (66-fold) if one charge is moving per transport cycle. TPP+ has been shown to equilibrate across membranes in an unmediated mode in response to electrical potentials and has been used to estimate their magnitudes (29). Here, TPP+ accumulates despite the fact that the potential is positive inside, suggesting that under these conditions the EmrE mediated flux is much faster than the unmediated one. The above findings suggest that, by definition, TPP+ transport is electrogenic, and the stoichiometry of the exchange reaction H+/TPP+ is therefore higher than 1.



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  		FIG. 5.
{Delta}{psi} drives uptake of TPP+ but not of MV2+ into proteoliposomes reconstituted with purified EmrE. Thiocyanate-loaded EmrE proteoliposomes (3 µl) were diluted into a thiocyanate-free medium containing [3H]TPP+ (black squares) or [14C]MV2+ (white squares), and radioactivity was incorporated at various time periods was measured as described under "Experimental Procedures." The values obtained with proteoliposomes diluted into a thiocyanate-containing medium were subtracted from all experimental points. Error bars are not seen because they are smaller than the symbols.

 
On the other hand, under the same conditions, EmrE does not catalyze MV2+ uptake (Fig. 5, white squares). These results indicate that unlike with the monovalent substrate TPP+, {Delta}{psi} cannot drive transport of the divalent substrate MV2+, whereas {Delta}pH drives transport of both substrates. Again, by definition we conclude that MV2+ transport is electroneutral, and therefore the stoichiometry of the exchange reaction H+/MV2+ is 2.

The Protonophore CCCP Affects TPP+ but Not MV2+ Uptake into Proteoliposomes—Another way to determine whether TPP+ transport by EmrE is electrogenic and MV2+ transport is electroneutral is by manipulation of gradients with ionophores. The equilibrium equation for a substrate-proton exchange reaction is given by Equation 1,

(Eq. 1)
where RT and F are physical constants, S and H denote the substrate and proton concentration in the inner (i) and outer (o) compartment, nH stands for the stoichiometry H+/substrate, and Z is the charge of the substrate (s) or the proton (H).

Thus, for a divalent substrate, transport will be electroneutral when nH = 2. In other words, under these conditions the membrane potential will not affect transport. When a pH gradient is generated (for example by an ammonium diffusion gradient) the addition of a protonophore such as carbonylcyanide m-chlorophenylhydrazone (CCCP) will generate a proton diffusion membrane potential equal to {Delta}pH but of opposite magnitude. The uptake of MV2+ into EmrE proteoliposomes was not affected by the addition of CCCP (Fig. 6A). Allowing the downhill movement of H+ by the addition of the K+ ionophore valinomycin, together with CCCP, discharged the {Delta}pH and inhibited MV2+ uptake (Fig. 6A). Similarly, the addition of the ionophore nigericin, which exchanges H+ and K+ ions, dissipates the pH gradient and inhibits MV2+ uptake.



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  		FIG. 6.
The effect of ionophores on TPP+ and MV2+ uptake. A, ammonium-loaded EmrE proteoliposomes (3 µl) were diluted into ammonium-free medium to generate a pH gradient. In addition the medium contained (A) [14C]MV2+ or (B) [3H]TPP+. Ionophores were added where indicated to the following concentrations: CCCP, 5 µM; valinomycin, 100 nM; nigericin, 15 µM. In C, the liposomes used are without protein. In A the reaction was terminated after 5 min, and in B and C the reaction was terminated after 1 min as described under "Experimental Procedures."

 
As shown by this equation, when the transport is electrogenic, transport will depend on with a bias on each component depending on the stoichiometry. Thus when nH = 2, the equilibrium equation for a substrate-proton exchange reaction is given by Equation 2.

(Eq. 2)

The results in Fig. 6B show that the accumulation of TPP+ into EmrE proteoliposomes was significantly albeit not fully reduced upon the addition of the protonophore CCCP. TPP+ uptake is inhibited when a combination of CCCP and valinomycin (Fig. 6B) or the ionophore nigericin (data not shown), which collapse under these conditions, are added. That CCCP does not fully inhibit TPP+ uptake is explained by the fact that, as shown previously, TPP+ accumulates inside liposomes as dictated by the membrane potential (inside negative) (29). This is illustrated in Fig. 6C where liposomes that do not contain protein cannot accumulate TPP+ when a pH gradient is generated. However, after the addition of CCCP (diffusion potential negative inside) TPP+ accumulates to levels similar to those observed in EmrE containing proteoliposomes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
EmrE transports a wide variety of aromatic cations. A comparison between the uptake of radiolabeled monovalent and divalent substrates into EmrE proteoliposomes was conducted under conditions where the driving forces were either a proton gradient or a transmembrane electrical potential. The results demonstrate that both forces drive the uptake of the monovalent substrate TPP+, but only the proton gradient can drive uptake of the divalent substrate MV2+. We conclude that the transport of monovalent substrates is electrogenic whereas the transport of the divalent ones is electroneutral. The difference in the transport modes is most likely because of the fact that the transporter uses the same proton/substrate stoichiometry with all the substrates. Because the valence of the substrates differs, different amounts of charge are translocated in one transport cycle. The findings imply that EmrE exchanges two protons per substrate.

High affinity ligand binding studies, functional complementation, and negative dominance studies all suggested that EmrE functions as an oligomer, but the size of the functional oligomer was not definitely established (11, 30, 31). Structural evidence from two-dimensional crystals led to the proposal that the minimal functional unit of EmrE was likely to be a dimer (32). This was confirmed by the determination of the three-dimensional structure of EmrE with TPP+ bound at 7.5 Å resolution by electron cryomicroscopy of two-dimensional crystals (33). The minimal structural unit is an asymmetric homodimer composed of eight transmembrane {alpha}-helices, i.e. four helices from each monomer, with density for TPP+ in a binding chamber formed from six of the 8 {alpha}-helices, confirming the suggestion that TPP+ binds near the center of the dimer (33). However, the two-dimensional crystals do not rule out the existence of higher order oligomers. In fact, co-expression of two plasmids in a cell free system allowed demonstration of functional complementation, and pull-down experiments confirmed that the basic functional unit is the dimer (34). An additional weaker interaction between dimers detected using cross-linking implies the existence of a dimer of dimers (34). In addition, in a model based on three-dimensional crystal diffracting to 3.8 Å, the protein is suggested to be a dimer of dimers (35).

The evidence presented in this study shows that the stoichiometry of the exchange reaction is two protons per substrate. Previous results showed that substrate binding to detergent-solubilized EmrE induces the release of almost one proton per EmrE monomer (14). Taking these two results together, the simplest conclusion is that the functional transport unit is the dimer. In the model mentioned above (35), the tetramer is the basic unit implying that substrate binding to EmrE would induce the release of 0.5 proton per EmrE monomer, a suggestion that is not borne out by experimental evidence (35).

Detergent-solubilized EmrE binds TPP+ with high affinity, and the study of this reaction has provided important information about the mechanism of catalysis by EmrE (11, 12). The measurements of ligand binding in detergent were validated by measurements of the pH profile of MV2+ transport in proteoliposomes reconstituted with purified EmrE (36). The finding that the protein binds TPP+ also when it is embedded in the phospholipid bilayer and that TPP+ is not only a ligand but also a bona fide substrate of EmrE provides a more direct substantiation of the binding measurements with detergent-solubilized protein. Transport is easily detected under the conditions used despite the fact that TPP+ is a lipophilic cation with a relatively high permeability through biological membranes. Up to now, we have not been able to show a significant resistance to TPP+ in intact cells expressing EmrE (17).2 The direct demonstration of transport in this work and the well known fact that TPP+ is a permeant cation that equilibrates with the membrane potential suggest that the lack of resistance is caused by the fact that the efflux rates catalyzed by EmrE in intact cells (with very low expression) were in the same order of magnitude of the passive uptake of TPP+.

We demonstrated here that the sensitivity of E. coli cells that express EmrE to monovalent drugs ethidium and acriflavine is much less affected by the rise in the external pH compared with the sensitivity to the divalent drug MV2+. In growing E. coli cells remains relatively constant at external pH values ranging from 5.0–8.0, whereas the electrical ({Delta}{psi}) and chemical ({Delta}pH) components interconvert. Because internal pH is kept constant at 7.6 as external pH is raised, {Delta}pH (inside alkaline) drops and even becomes inverted (inside acid) above pH 7.6, whereas {Delta}{psi} (inside negative) increases (25, 37). We conclude that the difference in the sensitivity results from the fact that only the electrogenic exchange of the monovalent substrates utilizes the membrane potential, whereas there is no driving force for the removal of divalent substrates at the high pH. Even though the remains relatively constant through the pH range tested, the sensitivity to the monovalent substrates increases at alkaline pH. As discussed above (Equation 2), this is because of the fact that when the stoichiometry H+/substrate is higher than 1, a decrease in {Delta}pH has a stronger effect on the substrate gradient that can be generated than the equivalent increase of {Delta}{psi}. Interestingly, the sensitivity of the cells that do not express EmrE increases at the alkaline pH values, again in a more pronounced form for the divalent MV2+. This may be caused by increased entry of the compounds because of the higher membrane potential, increased toxicity, or a reduced ability of the "housekeeping" multidrug transporters to remove the cations. The latter can be due again to the fact that also other transporters may transport divalent cations electrogenically and monovalents electroneutrally. Several other antibiotics and toxins seem to be more effective at alkaline pH (20, 38, 39). The ability to transport substrates with different valence has been reported for other bacterial secondary multidrug transporters; for example, QacA from Staphylococcus aureus confers resistance against a wide range of mono- and divalent lipophilic cations (16), and E. coli MdfA confers resistance against monovalent lipophilic cations and neutral compounds (15). As already mentioned, MdfA exchanges neutral compounds as chloramphenicol and thiamphenicol in an electrogenic mode, whereas it exchanges monovalent cationic substrates in an electroneutral mode (20).

These findings suggest a general property of multidrug transporters that recognize substrates with different valences and transport them in different modes. This could provide a novel strategy to overcome some of the drug resistance infections in the clinic because we could in theory engineer conditions where the MDTs are not efficient (for example whenever it is possible to raise the local pH of the environment) or develop new compounds that, under physiological conditions, will be poor substrates of the MDTs.


   FOOTNOTES
 
* This work was supported by Grant NS16708 from the National Institutes of Health and Grant 463/00 from the Israel Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} Recipient of a Yeshaya Horowitz Foundation fellowship. Back

§ To whom correspondence should be addressed. Tel.: 972-2-6585992; Fax: 972-2-5634625; E-mail: Shimon.Schuldiner{at}huji.ac.il.

1 The abbreviations used are: MDT, multidrug transporter; SMR, small multidrug resistance; MV2+, methyl viologen; TPP+, tetraphenylphosphonium; EmrE-His, EmrE tagged with Myc epitope and six His residues; DCCD, N,N'-dicyclohexylcarbodiimide; SCN–, thiocyanate; CCCP, carbonylcyanide m-chlorophenylhydrazone; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. Back

2 D. Rotem and S. Schuldiner, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Samanta Jamszon for performing some of the experiments and Misha Soskine for providing purified EmrE for the reconstitution.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Paulsen, I. T., Brown, M. H., and Skurray, R. A. (1996) Microbiol. Rev. 60, 575–608[Abstract/Free Full Text]
  2. Van Bambeke, F., Balzi, E., and Tulkens, P. M. (2000) Biochem. Pharmacol. 60, 457–470[CrossRef][Medline] [Order article via Infotrieve]
  3. Nikaido, H. (1994) Science 264, 382–388[Abstract/Free Full Text]
  4. Chung, Y. J., and Saier, M. H., Jr. (2001) Curr. Opin. Drug. Discovery Devel. 4, 237–245
  5. Paulsen, I. T., Skurray, R. A., Tam, R., Saier, M. H., Jr., Turner, R. J., Weiner, J. H., Goldberg, E. B., and Grinius, L. L. (1996) Mol. Microbiol. 19, 1167–1175[Medline] [Order article via Infotrieve]
  6. Schuldiner, S., Granot, D., Mordoch, S. S., Ninio, S., Rotem, D., Soskin, M., Tate, C. G., and Yerushalmi, H. (2001) News Physiol. Sci. 16, 130–134[Abstract/Free Full Text]
  7. Schuldiner, S., Granot, D., Steiner, S., Ninio, S., Rotem, D., Soskin, M., and Yerushalmi, H. (2001) J. Mol. Microbiol. Biotechnol. 3, 155–162[Medline] [Order article via Infotrieve]
  8. Ninio, S., Rotem, D., and Schuldiner, S. (2001) J. Biol. Chem. 276, 48250–48256[Abstract/Free Full Text]
  9. Ninio, S., and Schuldiner, S. (2003) J. Biol. Chem. 278, 12000–12005[Abstract/Free Full Text]
  10. Yerushalmi, H., Lebendiker, M., and Schuldiner, S. (1995) J. Biol. Chem. 270, 6856–6863[Abstract/Free Full Text]
  11. Muth, T. R., and Schuldiner, S. (2000) EMBO J. 19, 234–240[CrossRef][Medline] [Order article via Infotrieve]
  12. Yerushalmi, H., and Schuldiner, S. (2000) FEBS Lett. 476, 93–97[CrossRef][Medline] [Order article via Infotrieve]
  13. Yerushalmi, H., and Schuldiner, S. (2000) Biochemistry 39, 14711–14719[CrossRef][Medline] [Order article via Infotrieve]
  14. Soskine, M., Adam, Y., and Schuldiner, S. (2004) J. Biol. Chem. 279, 9951–9955[Abstract/Free Full Text]
  15. Bibi, E., Adler, J., Lewinson, O., and Edgar, R. (2001) J. Mol. Microbiol. Biotechnol. 3, 171–177[Medline] [Order article via Infotrieve]
  16. Brown, M. H., and Skurray, R. A. (2001) J. Mol. Microbiol. Biotechnol. 3, 163–170[Medline] [Order article via Infotrieve]
  17. Nishino, K., and Yamaguchi, A. (2001) J. Bacteriol. 183, 5803–5812[Abstract/Free Full Text]
  18. Ueda, K., Taguchi, Y., and Morishima, M. (1997) Semin. Cancer Biol. 8, 151–159[CrossRef][Medline] [Order article via Infotrieve]
  19. Schumacher, M. A., and Brennan, R. G. (2002) Mol. Microbiol. 45, 885–893[CrossRef][Medline] [Order article via Infotrieve]
  20. Lewinson, O., Adler, J., Poelarends, G. J., Mazurkiewicz, P., Driessen, A. J., and Bibi, E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 1667–1672[Abstract/Free Full Text]
  21. Yanish-Perron, C., Viera, J., and Messing, J. (1985) Gene (Amst.) 33, 103–199[CrossRef][Medline] [Order article via Infotrieve]
  22. Goldberg, E. B., Arbel, T., Chen, J., Karpel, R., Mackie, G. A., Schuldiner, S., and Padan, E. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 2615–2619[Abstract/Free Full Text]
  23. Tabor, S., and Richardson, C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1074–1078[Abstract/Free Full Text]
  24. Gerchman, Y., Olami, Y., Rimon, A., Taglicht, D., Schuldiner, S., and Padan, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1212–1216[Abstract/Free Full Text]
  25. Zilberstein, D., Schuldiner, S., and Padan, E. (1979) Biochemistry 18, 669–673[CrossRef][Medline] [Order article via Infotrieve]
  26. Yerushalmi, H., Mordoch, S. S., and Schuldiner, S. (2001) J. Biol. Chem. 276, 12744–12748[Abstract/Free Full Text]
  27. Rudnick, G. (1998) Methods Enzymol. 296, 233–247[CrossRef][Medline] [Order article via Infotrieve]
  28. Schuldiner, S., Shirvan, A., and Linial, M. (1995) Physiol. Rev. 75, 369–392[Free Full Text]
  29. Schuldiner, S., and Kaback, H. R. (1975) Biochemistry 14, 5451–5461[CrossRef][Medline] [Order article via Infotrieve]
  30. Rotem, D., Sal-man, N., and Schuldiner, S. (2001) J. Biol. Chem. 276, 48243–48249[Abstract/Free Full Text]
  31. Yerushalmi, H., Lebendiker, M., and Schuldiner, S. (1996) J. Biol. Chem. 271, 31044–31048[Abstract/Free Full Text]
  32. Tate, C. G., Kunji, E. R., Lebendiker, M., and Schuldiner, S. (2001) EMBO J. 20, 77–81[CrossRef][Medline] [Order article via Infotrieve]
  33. Ubarretxena-Belandia, I., Baldwin, J. M., Schuldiner, S., and Tate, C. G. (2003) EMBO J. 22, 6175–6181[CrossRef][Medline] [Order article via Infotrieve]
  34. Elbaz, Y., Steiner-Mordoch, S., Danieli, T., and Schuldiner, S. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 1519–1524[Abstract/Free Full Text]
  35. Ma, C., and Chang, G. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 2852–2857[Abstract/Free Full Text]
  36. Yerushalmi, H., and Schuldiner, S. (2000) J. Biol. Chem. 275, 5264–5269[Abstract/Free Full Text]
  37. Padan, E., and Schuldiner, S. (1987) J. Membr. Biol. 95, 189–198[CrossRef][Medline] [Order article via Infotrieve]
  38. Minakami, H., Kitzler, J. W., and Fridovich, I. (1990) J. Bacteriol. 172, 691–695[Abstract/Free Full Text]
  39. Eisenberg, E. S., Mandel, L. J., Kaback, H. R., and Miller, M. H. (1984) J. Bacteriol. 157, 863–867[Abstract/Free Full Text]

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